CN107715170B

CN107715170B - 3D polypyrrole chitosan gelatin composite conductive material and preparation method thereof

Info

Publication number: CN107715170B
Application number: CN201711223778.9A
Authority: CN
Inventors: 何生; 阎长平; 李仁科; 姜增誉; 李健丁; 宋慧芳
Original assignee: First Hospital of Shanxi Medical University
Current assignee: First Hospital of Shanxi Medical University
Priority date: 2017-11-28
Filing date: 2017-11-28
Publication date: 2020-11-10
Anticipated expiration: 2037-11-28
Also published as: CN107715170A

Abstract

The invention relates to a preparation method of a 3D polypyrrole chitosan gelatin composite conductive material, which comprises the steps of firstly, reacting chitosan and pyrrole serving as raw materials in the presence of a single electron reducing agent ferric trichloride to generate a polypyrrole-chitosan polymer; and then, crosslinking the polypyrrole-chitosan polymer in the pores of the gelatin sponge sheet in the presence of a crosslinking agent glutaraldehyde to form the 3D polypyrrole-chitosan gelatin composite electric conduction material with a porous structure. The 3D polypyrrole chitosan gelatin composite conductive material prepared by the method has the advantages of good mechanical strength, good biocompatibility, high conductivity, low impedance and small voltage loss, and can accelerate Ca among myocardial cells²⁺And (4) signal transmission. In-vitro evaluation experiments prove that the 3D polypyrrole chitosan gelatin composite conductive material has feasibility and effectiveness when being used as a repairing material to replace the defect of the full-layer right ventricular outflow tract in the heart of a rat.

Description

3D polypyrrole chitosan gelatin composite conductive material and preparation method thereof

Technical Field

The invention belongs to the technical field of materials, and particularly relates to a 3D polypyrrole chitosan gelatin composite conductive material and a preparation method thereof.

Background

With the development of the economic society, the birth rate of children suffering from Congenital Heart Disease (CHD) is already obviously reduced; however, research shows that the CHD yield is as high as 0.9 percent in Asian regions including China, the CHD is one of diseases seriously threatening the healthy growth of children, and heavy burden is brought to individuals, families and society. In recent years, myocardial infarction causes death of a large number of myocardial cell fibers to form scars and fibrosis, destroys the original point activity conduction path of the heart, not only causes reduction of systolic and diastolic functions of the heart, but also causes abnormal electrical activity of the heart, such as delayed conduction and various types of arrhythmia. Reconstructing scar zone conduction can synchronize the surviving myocardium. Congenital heart disease is one of the leading causes of infant death.

Cardiac tissue engineering has attracted considerable attention as a means of repairing cardiac defects. Many degradable biomaterials (e.g., alginic acid, collagen, gelatin sponge, and polyglycolic acid) are used to repair defects or stabilize infarcted areas of the heart and prevent expansion of the heart. Earlier researches show that in an animal myocardial infarction model, gelatin sponge is used for loading myocardial cells for patch transplantation, and the gelatin sponge is proved to be capable of supporting cell survival and promoting local angiogenesis; the alginic acid porous patch is also used for experimental study of the myocardial infarction; 3D polyglycolic acid as a biocompatible material can support the division and proliferation of embryonic stem cells to cardiomyocyte-like cells. Patients with mild to moderate ventricular dysfunction may benefit from cytokine injection, cell patches, hydrogel injection, or surface reinforcement of the myocardial infarction site. However, these biomaterials are poor in conductivity or insulators, which limits their applications, and there is an urgent need to develop new materials to solve the above problems.

The conductive biomaterial is a special conductive polymeric material with metal ion conductivity; many conductive polymer materials are applied to biological tissue engineering, and Ppy is the most studied conductive polymer and has good conductivity and biocompatibility. The 3D Ppy-PLGA conductive composite material synthesized by scholars is applied to the study of the myocardial progenitor cells and the IPS cells, has good biocompatibility and conductivity, and can support the proliferation and differentiation capacity of the myocardial progenitor cells and the IPS cells to the myocardium.

However, few studies have investigated the feasibility of 3D composite conductive patch materials for cardiac tissue engineering to improve cardiomyocyte conductivity and cardiac electrical pulse propagation through their electrochemical properties.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a 3D polypyrrole chitosan gelatin composite conductive material and a preparation method thereof. The 3D polypyrrole chitosan gelatin composite conductive material prepared by the method has good biocompatibility, higher conductivity and lower impedance, and can accelerate calcium signal transmission among myocardial cells.

The technical scheme adopted by the invention is as follows:

(1) preparing an acetic acid solution, adding chitosan into the acetic acid solution, controlling the mass ratio of acetic acid to chitosan in the acetic acid solution to be 0.4:1-0.6:1, and fully and uniformly mixing under the stirring condition to obtain a chitosan acid solution;

(2) adding pyrrole into the chitosan acid solution obtained in the step (1), controlling the mass ratio of chitosan to pyrrole in the chitosan acid solution to be 1:0.03-1:0.4, and fully and uniformly mixing under the stirring condition to obtain a chitosan-pyrrole mixed solution;

(3) preparing a ferric trichloride solution, slowly dripping the ferric trichloride solution into the chitosan-pyrrole mixed solution in a dark room under the stirring condition, and controlling the mass ratio of ferric trichloride to pyrrole in the ferric trichloride solution to be 1:0.2-1: 0.4; after the dripping is finished, continuously stirring the mixture until the mixture fully reacts to obtain black polypyrrole-chitosan polymer solution;

(4) putting the polypyrrole-chitosan polymer solution into a reverse osmosis type dialysis bag, dialyzing by adopting PBS buffer solution to remove redundant iron ions and pyrrole, and then adjusting the pH value of the polypyrrole-chitosan polymer solution to 5.8-6.5 by adopting disodium glycerophosphate for later use;

(5) and (3) adding gelatin sponge sheets into the polypyrrole-chitosan polymer solution after the pH is adjusted in the step (4), fully mixing uniformly, adding glutaraldehyde, controlling the volume ratio of the glutaraldehyde to the polypyrrole-chitosan polymer solution to be 1:250-1:1000, fully reacting, and sequentially dehydrating and freeze-drying to obtain the 3D polypyrrole-chitosan gelatin composite conductive material.

In the step (1), the concentration of the acetic acid solution is 0.17-0.18 mol/L.

In the step (1), the stirring speed is 300-500rpm, and the stirring time is 2-4 h.

In the step (2), the stirring speed is 300-500rpm, and the stirring time is 8-12 h.

In the step (3), the concentration of the ferric trichloride solution is 0.067-0.67 mol/L.

In the step (3), the dropping speed of the ferric trichloride solution is 0.3-0.6 ml/h.

In the step (4), the PBS buffer solution is 0.05 XPBS-0.2 XPPBS.

In the step (4), the mass concentration of the disodium glycerophosphate is 45-55%.

In the step (5), the specification of the gelatin sponge sheet is 10mm by 3mm by 60mm, and the mass concentration of the glutaraldehyde is 0.4-4%;

the dehydration is alcohol gradient dehydration (alcohol gradient concentration is 30% -50% -70% -80% -90% -100% in sequence, each time is 3min, and the time is 2 times), and the freeze-drying time is 12-24 h.

The 3D polypyrrole chitosan gelatin composite conductive material prepared by the method.

The invention has the beneficial effects that:

the preparation method of the 3D polypyrrole chitosan gelatin composite conductive material provided by the invention comprises the steps of firstly, reacting chitosan and pyrrole serving as raw materials in the presence of a single electron reducing agent ferric trichloride to generate a polypyrrole-chitosan polymer; and then, crosslinking the polypyrrole-chitosan polymer in the pores of the gelatin sponge sheet in the presence of a crosslinking agent glutaraldehyde to form the 3D polypyrrole-chitosan gelatin composite electric conduction material with a porous structure. The 3D polypyrrole chitosan gelatin composite conductive material prepared by the method has the advantages of good mechanical strength, good biocompatibility, high conductivity, low impedance and small voltage loss, and can accelerate Ca among myocardial cells²⁺And (4) signal transmission. In vitro evaluation experiments prove that the 3D polypyrrole chitosan amineThe glue composite conductive material has feasibility and effectiveness as a repairing material in rat hearts to replace full thickness Right Ventricular Outflow Tract (RVOT) defects.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a diagram showing the reaction mechanism of chitosan and pyrrole to form polypyrrole chitosan;

FIG. 2 is a diagram of the synthesis mechanism of the 3D polypyrrole chitosan gelatin composite conductive material formed by crosslinking polypyrrole chitosan into gelatin pores;

FIGS. 3A-3C are infrared spectra of pyrrole, chitosan, polypyrrole chitosan, respectively;

FIGS. 4A-4C are SEM images of composite electric conductive material of gelatin, chitosan gelatin and polypyrrole chitosan gelatin, respectively;

FIG. 5 is a graph showing the stress variation of a composite conductive material of gelatin, chitosan gelatin and polypyrrole chitosan gelatin;

FIG. 6 is a comparison graph of the fracture stress statistical analysis of the composite conductive material of gelatin, chitosan gelatin and polypyrrole chitosan gelatin;

FIG. 7 is a comparative analysis chart of biocompatibility of gelatin (Gel), chitosan gelatin (Chi-Gel), polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel);

FIG. 8A is a diagram of the cycle I-V of gelatin (Gel), chitosan gelatin (Chi-Gel), polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel);

FIG. 8B is a graph showing the comparison of the electrical conductivity of gelatin (Gel), chitosan gelatin (Chi-Gel), and polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel);

FIG. 9 is a graph comparing the statistical results of the voltage recorded after passing the electric signal for gelatin (Gel), chitosan gelatin (Chi-Gel), polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel);

FIG. 10 shows Ca between NRVMs on two regions of interest (ROI #1, ROI #2) on Gel, Chi-Gel, Ppy-Chi-Gel²⁺A signal transmission speed statistical analysis comparison graph;

FIG. 11 is an echocardiogram 7 days after observation of Ppy-Chi-Gel patch by cardiac ultrasound;

FIG. 12A shows the patch area change after 7 days of Gel, Chi-Gel, Ppy-Chi-Gel transplantation;

FIG. 12B shows the patch area change after transplanting Gel, Chi-Gel, Ppy-Chi-Gel for 28 days;

FIG. 12C is the statistical results of the cross-sectional thickness of the patch after transplanting Gel, Chi-Gel, Ppy-Chi-Gel for 28 days;

FIG. 13 is a comparison graph of statistical analysis of electrical signal conduction velocity between Gel, Chi-Gel, and Ppy-Chi-Gel in defective myocardium.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Example 1

The embodiment provides a preparation method of a 3D polypyrrole chitosan gelatin composite electric conduction material, which comprises the following steps:

(1) preparing an acetic acid solution with the concentration of 0.172mol/L, adding chitosan into the acetic acid solution, controlling the mass ratio of acetic acid to chitosan in the acetic acid solution to be 0.525:1, and stirring at 400rpm for 4 hours to fully and uniformly mix to obtain a chitosan acid solution;

(2) adding pyrrole into the chitosan acid solution obtained in the step (1), controlling the mass ratio of chitosan to pyrrole in the chitosan acid solution to be 1:0.294, and stirring at 400rpm for 12 hours to fully and uniformly mix to obtain a chitosan-pyrrole mixed solution;

(3) preparing a ferric trichloride solution with the concentration of 0.67mol/L, slowly dripping the ferric trichloride solution into the chitosan-pyrrole mixed solution at the dripping speed of 0.5ml/h in a dark room at the temperature of 4 ℃ under the stirring condition, and controlling the mass ratio of ferric trichloride to pyrrole in the ferric trichloride solution to be 1: 0.3; after the dripping is finished, stirring for 48 hours continuously for full reaction to obtain black polypyrrole-chitosan polymer solution (Ppy-Chi), wherein a reaction mechanism diagram is shown in figure 1;

(4) the polypyrrole-chitosan polymer solution was placed in a 12-14kD reverse osmosis dialysis bag using 0.1 XPBS (1L of Na in solution)₂HPO₄0.142g, KH₂PO₄0.027g, NaCl 0.8g, KCl 0.02g) buffer solution to remove redundant iron ions and pyrrole, and then adopting disodium glycerophosphate with the mass concentration of 50% to adjust the pH value of the polypyrrole-chitosan polymer solution to 6 for later use;

(5) adding gelatin sponge sheets with the specification of 10mm x 3mm x 60mm into the polypyrrole-chitosan polymer solution after the pH is adjusted in the step (4), fully and uniformly mixing, adding glutaraldehyde with the mass concentration of 4%, controlling the volume ratio of the glutaraldehyde to the polypyrrole-chitosan polymer solution to be 1:250, fully reacting, performing gradient dehydration by using alcohol (the gradient concentration of the alcohol is 30% -50% -70% -80% -90%, eluting for 3min each time, eluting for 2 times altogether), and freeze-drying for 24 hours to obtain the 3D polypyrrole-chitosan gelatin composite conductive material (Ppy-Chi-Gel), wherein the synthetic mechanism diagram is shown in fig. 2.

Example 2

(1) preparing an acetic acid solution with the concentration of 0.17mol/L, adding chitosan into the acetic acid solution, controlling the mass ratio of acetic acid to chitosan in the acetic acid solution to be 0.4:1, and stirring at 300rpm for 4 hours to fully and uniformly mix to obtain a chitosan acid solution;

(2) adding pyrrole into the chitosan acid solution obtained in the step (1), controlling the mass ratio of chitosan to pyrrole in the chitosan acid solution to be 1:0.03, and stirring at 300rpm for 12 hours to fully and uniformly mix to obtain a chitosan-pyrrole mixed solution;

(3) preparing a ferric trichloride solution with the concentration of 0.067mol/L, slowly dripping the ferric trichloride solution into the chitosan-pyrrole mixed solution at the dripping speed of 0.3ml/h in a dark room at the temperature of 4 ℃ under the stirring condition, and controlling the mass ratio of ferric trichloride to pyrrole in the ferric trichloride solution to be 1: 0.2; after the dripping is finished, stirring for 24 hours continuously for full reaction to obtain black polypyrrole-chitosan polymer solution (Ppy-Chi);

(4) putting the polypyrrole-chitosan polymer solution into a 12-14kD reverse osmosis dialysis bag, dialyzing by using 0.05 times PBS buffer solution to remove redundant iron ions and pyrrole, and then adjusting the pH value of the polypyrrole-chitosan polymer solution to 5.8 by using 45% of disodium glycerophosphate for later use;

(5) and (3) adding gelatin sponge sheets with the specification of 10mm x 3mm x 60mm into the polypyrrole-chitosan polymer solution after the pH is adjusted in the step (4), fully and uniformly mixing, adding glutaraldehyde with the mass concentration of 0.4%, controlling the volume ratio of the glutaraldehyde to the polypyrrole-chitosan polymer solution to be 1:250, fully reacting, performing gradient dehydration by using alcohol, and freeze-drying for 12h to obtain the 3D polypyrrole-chitosan gelatin composite conductive material (Ppy-Chi-Gel).

Example 3

(1) preparing an acetic acid solution with the concentration of 0.18mol/L, adding chitosan into the acetic acid solution, controlling the mass ratio of acetic acid to chitosan in the acetic acid solution to be 0.6:1, and stirring for 2 hours at 500rpm for fully and uniformly mixing to obtain a chitosan acid solution;

(2) adding pyrrole into the chitosan acid solution obtained in the step (1), controlling the mass ratio of chitosan to pyrrole in the chitosan acid solution to be 1:0.04, and stirring at 500rpm for 8 hours to fully and uniformly mix to obtain a chitosan-pyrrole mixed solution;

(3) preparing a ferric trichloride solution with the concentration of 0.67mol/L, slowly dripping the ferric trichloride solution into the chitosan-pyrrole mixed solution at the dripping speed of 0.3ml/h in a dark room at the temperature of 4 ℃ under the stirring condition, and controlling the mass ratio of ferric trichloride to pyrrole in the ferric trichloride solution to be 1: 0.4; after the dripping is finished, stirring for 48 hours continuously for full reaction to obtain black polypyrrole-chitosan polymer solution (Ppy-Chi);

(4) putting the polypyrrole-chitosan polymer solution into a 12-14kD reverse osmosis dialysis bag, dialyzing by using 0.2 XPBS (phosphate buffer solution) to remove redundant iron ions and pyrrole, and then adjusting the pH value of the polypyrrole-chitosan polymer solution to 6.5 by using 65% of disodium glycerophosphate for later use;

(5) and (3) adding gelatin sponge sheets with the specification of 10mm x 3mm x 60mm into the polypyrrole-chitosan polymer solution after the pH is adjusted in the step (4), fully and uniformly mixing, adding glutaraldehyde with the mass concentration of 4%, controlling the volume ratio of the glutaraldehyde to the polypyrrole-chitosan polymer solution to be 1:250, fully reacting, performing gradient dehydration by using alcohol, and freeze-drying for 24 hours to obtain the 3D polypyrrole-chitosan gelatin composite conductive material (Ppy-Chi-Gel).

Comparative example

This comparative example provides a chitosan gelatin as a control, prepared as follows:

after the chitosan acidic solution was prepared in example 1, the solution was dialyzed directly without adding ferric chloride solution, and then adjusted to pH 6, glutaraldehyde was added in the same amount as in example 1, and other steps were performed, and chitosan gelatin (Chi-Gel) was synthesized as a control.

Examples of the experiments

1. Infrared spectroscopic analysis of polypyrrole chitosan polymers

FIG. 3A is an infrared spectrum of 98% pyrrole at 942cm^-1And 1014cm^-1The characteristic absorption wavelength of the N-C bond can be observed; at 1669cm^-1The N-H bond frequency was observed at 3389cm, due to the presence of water therein^-1The O-H bond frequency can be observed(ii) a FIG. 3B is an infrared spectrum of chitosan at 977cm^-1And 1088cm^-1The characteristic absorption peaks of C-O and C-O-C, which are the main chemical bond components of Chi, can be observed to be 1638cm^-1And 3304cm^-1(ii) a FIG. 3C is an infrared spectrum of Ppy-Chi (polypyrrole chitosan polymer) at 950cm^-1、1064cm^-1The characteristic frequency of both N-C, C-O-C was observed at 1654cm^-1It can be observed that the presence of N-H bonds, due to the interaction of functional groups and chemical bonds, is also reflected in this test, as the same functional groups and chemical bonds have some translation in different materials.

2. Ultrastructure analysis of gelatin (Gel), chitosan gelatin (Chi-Gel) and polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel)

Dehydrating the three materials (Gel, Chi-Gel, Ppy-Chi-Gel) with gradient alcohol and liquid CO respectively₂After the critical point is dried, gold coating treatment and scanning electron microscope examination are carried out, the results are shown in fig. 4A-4C, and the results show that the surface pore sizes of the three materials of Gel, Chi-Gel and Ppy-Chi-Gel are different, the Chi-Gel and Ppy-Chi-Gel are smaller than the Gel, and more granular substances are covered on the surfaces of the materials of Chi-Gel and Ppy-Chi-Gel.

3. Mechanical strength analysis of gelatin (Gel), chitosan gelatin (Chi-Gel) and polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel)

As shown in FIG. 5, which is a typical stress variation force comparison graph of Gel, Chi-Gel and Ppy-Chi-Gel, it can be seen that the fracture stresses of Gel, Chi-Gel and Ppy-Chi-Gel are gradually increased, and respectively are 8.24 + -1.52 Kpa, 12.37 + -2.76 Kpa and 19.21 + -3.16 Kpa.

FIG. 6 shows a comparison graph of the fracture stress statistical analysis of Gel, Chi-Gel, Ppy-Chi-Gel.

4. Comparative analysis of biocompatibility of gelatin (Gel), chitosan gelatin (Chi-Gel) and polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel)

Whether cells can adhere to the surface of the material or not is used for evaluating the biocompatibility of the material, newly separated suckling mouse myocardial cells (NRVM) are inoculated on the surfaces of Gel, Chi-Gel and Ppy-Chi-Gel according to the same concentration, DF12 is cultured for 1, 3 and 5 days respectively, and cell nucleuses are marked by DAPI to detect the cell number; no significant difference in cell numbers was found among the three materials Gel, Chi-Gel, Ppy-Chi-Gel at observation points of 1, 3, and 5 days (as shown in FIG. 7).

5. Conductivity tests of gelatin (Gel), chitosan gelatin (Chi-Gel) and polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel)

The three materials Gel, Chi-Gel and Ppy-Chi-Gel are cut into cuboids with the same size, and the conductivity of the three materials is measured by a cyclic voltammetry method. The method specifically comprises the following steps: placing the material on a HP4155 test bench of a semiconductor tester, wherein the distance (D) between two probes is 7mm, and the circulating voltage is-5-5V; the slope is measured and the conductivity of the corresponding material is calculated by applying the formula 1/[2 π D (V/I) ].

FIG. 8A is a cyclic I-V diagram of three materials, namely Gel, Chi-Gel and Ppy-Chi-Gel, obtained by an HP4155 semiconductor tester, with the change of voltage, the Gel current is basically near 0 point and shows an insulator characteristic, the Chi-Gel and Ppy-Chi-Gel currents are gradually increased and show a semiconductor characteristic, and the peak value of the Ppy-Chi-Gel current is obviously higher than those of the Chi-Gel and the Gel.

FIG. 8B shows that the conductivity of the three materials Gel, Chi-Gel and Ppy-Chi-Gel is obviously different, the conductivity of the Gel is close to zero, the conductivity of the Chi-Gel is (4.78 +/-1.61) × 10-5S/cm, the conductivity of the Ppy-Chi-Gel is (12.49 +/-2.25) × 10-5S/cm, which is about 3 times of the Chi-Gel, and the newly synthesized Ppy-Chi-Gel material is proved to have higher conductivity.

6. Voltage loss test of gelatin (Gel), chitosan gelatin (Chi-Gel) and polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel)

In the series circuit, the larger the current passing through the resistor, the more the voltage is lost, and the smaller the measured voltage is, based on the principle, an electric signal (1mV, 80 times/min) with the same intensity is used for stimulating one side of Gel, Chi-Gel and Ppy-Chi-Gel, the voltage value is recorded by ECG on the opposite side, the voltage values of the three materials are compared, the resistance of the three materials is indirectly reflected to be different, and further the conductivity of the three materials is different.

FIG. 9 shows statistical results of the electric signals from the recording rate to the voltage through Gel, Chi-Gel, Ppy-Chi-Gel, wherein the recorded electric signals are close to 0 in the Gel material, the recorded voltage value is 0.13 + -0.03 mV in the Chi-Gel material, and the recorded voltage value is 0.34 + -0.13 mV in the Ppy-Chi-Gel material, which is obviously higher than the former two.

7. Ca among NRVM cells of gelatin (Gel), chitosan gelatin (Chi-Gel) and polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel)²⁺The conduction velocity.

Inoculating the newly separated NRVM cell on Gel, Chi-Gel and Ppy-Chi-Gel materials, culturing DF-12 for 5 days, and culturing intercellular Ca²⁺Ca for instantaneous signal transmission²⁺Marking by sensitive tracer Fluo-4AM, exciting by 488nm laser, recording in a microscope system by using an optical high-speed electron charge coupled device imaging (EMCCD) system, and calculating Ca between two interested areas by Image J software²⁺Instantaneous transfer speed.

FIG. 10 shows Ca between NRVMs on two regions of interest (ROI #1, ROI #2) on three materials Gel, Chi-Gel, Ppy-Chi-Gel²⁺Statistical analysis of signal Transmission Rate, Ca between Gel and Chi-Gel, NRVM²⁺The signal transduction rates averaged 20 mm/s, while the transduction rate on Ppy-Chi-Gel was 70 mm/s, the difference being statistically significant (P<0.01，n＝6)。

8. Application of gelatin (Gel), chitosan gelatin (Chi-Gel) and polypyrrole chitosan gelatin composite electric conduction material (Ppy-Chi-Gel) in right heart outflow tract defect model of rat and evaluation

8.1SD rat RVOT defect model and patch operation

The method of local freezing by using liquid nitrogen can cause transmural death of RVOT local myocardial tissues, then on the basis of peripheral purse string suture, the central necrotic myocardial tissues are reduced to form a defect with the diameter of 3mm, and then Gel, Chi-Gel and Ppy-Chi-Gel are sutured on the surface of the defect by using a surgical suture line to form a patch-like structure.

As shown in FIG. 11, the echocardiogram 7 days after the Ppy-Chi-Gel patch is observed in the cardiac ultrasound, the short axis level of the echocardiogram can clearly show the RVOT local myocardial tissue defect and the graft material on the surface, the arrow shows the RVOT defect position, the coil shows the position of the Ppy-Chi-Gel material, the LV left ventricle and the RV right ventricle.

8.2 degradation of Gel, Chi-Gel, Ppy-Chi-Gel in vivo

7 days and 28 days after the operation, respectively transplanting a certain amount of Gel, Chi-Gel and Ppy-Chi-Gel materials into an SD rat after the operation to separate the heart under the anesthesia state, stopping the heart in diastole by using cardioplegic arrest liquid, performing high-definition photographing by using a Canon camera according to the original proportion, and performing statistical analysis on the surface area of the materials through Image J.

FIG. 12A shows the surface area changes of Gel, Chi-Gel, Ppy-Chi-Gel after 7 days of transplantation, with no statistical difference (P >0.05) among three groups; FIG. 12B shows the surface area changes of Gel, Chi-Gel, Ppy-Chi-Gel after 28 days of transplantation, and the surface area of Ppy-Chi-Gel group is significantly larger than that of the other two groups (P < 0.01). FIG. 12B is a ring diagram of 2mm thick cardiac samples implanted 28 days after the implantation of Gel, Chi-Gel, Ppy-Chi-Gel, which is observed by covering the outer surface of the RVOT defect of the rat with Gel, Chi-Gel, Ppy-Chi-Gel materials, and observing the thickness variation and statistical analysis results of the material thickness of the local Gel, Chi-Gel, Ppy-Chi-Gel materials, and Ppy-Chi-Gel is thicker than the other two groups.

9. Research on electrical signal conduction velocity of gelatin (Gel), chitosan gelatin (Chi-Gel) and polypyrrole chitosan gelatin composite conductive material (Ppy-Chi-Gel) between defected myocardium

In vitro, we have demonstrated that Ppy-Chi-Gel accelerates intracellular Ca in NRVM cells²⁺Signal transmission; and (3) observing whether the Ppy-Chi-Gel material can accelerate the conduction speed of the electrical signals between the defected myocardium by using OPTICAL MAPPING. The method specifically comprises the following steps: gel, Chi-Gel, Ppy-Chi-Gel grafts were performed immediately on the RVOT defect model, hearts were isolated 28 days later and perfused with voltage sensitive dye, RVOT signal changes were recorded with ECCD and analyzed using the BrainVision 1508. The local electric signal conduction of the normal heart RVOT, RVOT-Gel, RVOT-Chi-Gel and RVOT-Ppy-Chi-Gel changes along with time, and the speed is respectively 0.92 +/-0.12 m/s, 0.58 +/-0.04 m/s, 0.66 +/-0.09 m/s and 0.82 +/-0.04 m/s. As shown in FIG. 13, the RVOT-Ppy-Chi-Gel local electrical signal conducted significantly faster than RVOT-Gel and RVOT-Chi-Gel (P)<0.05) without significant difference from the normal RVOT local electrical signal conduction velocity.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. A preparation method of a 3D polypyrrole chitosan gelatin composite electric conduction material is characterized by comprising the following steps:

2. The method for preparing a 3D polypyrrole chitosan gelatin composite electric conduction material according to claim 1, wherein in the step (1), the concentration of the acetic acid solution is 0.17-0.18 mol/L.

3. The method for preparing a 3D polypyrrole chitosan gelatin composite electric conduction material according to claim 1, wherein in the step (1), the stirring speed is 300-500rpm, and the stirring time is 2-4 h.

4. The method for preparing a 3D polypyrrole chitosan gelatin composite electrical conduction material according to claim 1, wherein in the step (2), the stirring speed is 300-500rpm, and the stirring time is 8-12 h.

5. The method for preparing a 3D polypyrrole chitosan gelatin composite electric conduction material according to claim 1, wherein in the step (3), the concentration of the ferric trichloride solution is 0.067-0.67 mol/L.

6. The method for preparing a 3D polypyrrole chitosan gelatin composite electric conduction material according to claim 1, characterized in that, in the step (3), the dropping speed of the ferric trichloride solution is 0.3-0.6 ml/h.

7. The method for preparing a 3D polypyrrole chitosan gelatin composite electric conduction material according to claim 1, wherein in the step (4), the PBS buffer solution is 0.05 x PBS-0.2 x PBS.

8. The method for preparing the 3D polypyrrole chitosan gelatin composite electric conduction material according to the claim 1, wherein in the step (4), the mass concentration of the disodium glycerophosphate is 45-55%.

9. The method for preparing a 3D polypyrrole chitosan gelatin composite electric conduction material according to claim 1, characterized in that, in the step (5), the gelatin sponge sheet has a specification of 10mm by 3mm by 60mm, and the mass concentration of the glutaraldehyde is 0.4-4%;

the dehydration is alcohol gradient dehydration, and the freeze-drying time is 12-24 h.

10. The 3D polypyrrole chitosan gelatin composite electric conductive material prepared by the method of any one of claims 1 to 9.